Method for controlling a fermentation process

Abstract

The present invention relates to a fermentation method for fermenting at least one microorganism, wherein the fermentation method comprises the steps of: (a) flowing a fermentation broth comprising at least one microorganism into a fermentation reactor; (b) supplying a carbon substrate to the fermentation reactor to dissolve or partially dissolve the gaseous carbon substrate in the fermentation broth; (c) supplying a nitrogen substrate to the fermentation reactor to dissolve or partially dissolve the gaseous nitrogen substrate in the fermentation broth; and (d) maintaining a nitrate concentration in the fermentation broth below 0.035 g / L and / or maintaining a nitrate concentration in the fermentation broth below 0.01 g nitrate / g biomass, wherein the at least one methanotrophic organism comprises at least one methanotrophic microorganism.

Description

[Technical field]

[0001] The present invention relates to fermentation methods and fermentation reactors for improving biomass production. In particular, the present invention relates to methods and fermentation reactors for fermenting methanotrophic organisms in which the concentration of nitrate is tightly controlled to optimize the fermentation process. [Background technology]

[0002] A nitrogen source, along with a carbon source, is essential for the growth of microorganisms during fermentation. Microorganisms need a nitrogen source to synthesize proteins, nucleic acids, and other cellular components.

[0003] Depending on the enzymatic capabilities of the microorganism, nitrogen may be provided as bulk proteins, such as soybean meal; as predigested polypeptides, such as peptone or tryptone; or as ammonia or nitrate. The choice of nitrogen source is important and may depend on the product being produced, as the cost of the nitrogen source is an important factor.

[0004] Even though the nitrogen source is an essential component for microbial growth, it is also known in the art that methanotrophic microorganisms are very sensitive to nitrogen loading, which may be affected by the form of the nitrogen source and the amount of the nitrogen source.

[0005] In the prior art it is speculated that this difference in ammonia and nitrite tolerance may be due to different affinities of, for example, the methane monooxygenase enzyme for the toxic effects of ammonia or nitrite.

[0006] Methane monooxygenase enzymes are responsible for conferring methanotrophy to methanotrophic microorganisms while simultaneously carrying out the oxidation of available nitrogen sources, resulting in a number of cometabolic by-products.

[0007] When growing methanotrophic microorganisms, such as M. capsulatus, nitrogen sources, such as ammonia, are readily oxidized by the methane monooxygenase of Methylococcus capsulatus, even at low extracellular concentrations, provided methane is not in large excess.

[0008] Ammonia is often used as the nitrogen source in fermentations to make cost-competitive single-cell protein (SCP) products from Methylococcus capsulatus fermentation. The solubility of ammonia in aqueous fermentation broth is orders of magnitude greater than the solubility of methane, which may be used as a carbon source, making ammonia oxidation a real challenge, even though the obvious and immediate problem of gas-to-liquid mass transfer is addressed by using appropriate reactor design.

[0009] Thus, improved fermentation methods and / or fermentation reactors would be advantageous, particularly more efficient and / or controlled fermentation methods and / or fermentation reactors where the nitrogen source is adjusted to improve production of methanotrophic biomass. Summary of the Invention

[0010] An object of the present invention therefore relates to an improved fermentation process for fermenting methanotrophic microorganisms, such as Methylococcus capsulatus.

[0011] In particular, it is an object of the present invention to provide a more efficient and / or controlled fermentation method and / or fermentation reactor in which the nitrogen source is adjusted to improve the production of methanotrophic biomass, and a fermentation method and / or fermentation reactor in which the level of the source material fed during fermentation is controlled to provide nutrients for the growth of microorganisms, e.g. methanotrophic microorganisms, while at the same time avoiding levels that create competitive inhibitors of methane consumption, thus solving the above-mentioned problems of the prior art.

[0012] Thus, one aspect of the present invention relates to a fermentation method for fermenting a fermentation broth comprising at least one microorganism in a fermentation reactor, the fermentation method comprising the steps of: a) feeding a carbon substrate to a fermentation reactor such that the carbon substrate is dissolved or partially dissolved in the fermentation broth; b) feeding the nitrogen substrate to a fermentation reactor so that the nitrogen substrate is dissolved or partially dissolved in the fermentation broth; and c) maintaining the nitrate concentration of the fermentation broth at less than 0.035 g / l and / or maintaining the nitrate concentration of the fermentation broth at less than 0.01 g nitrate / g biomass wherein the at least one microorganism comprises at least one methanotrophic microorganism.

[0013] Another aspect of the invention relates to a fermentation reactor comprising a loop section and an upper tank, said loop section comprising a downflow section connected to an upflow section via a U-section, said upper tank comprising: (i) a first outlet connecting the upper tank to the downflow portion of the loop portion and allowing the fermentation liquid present in the upper tank to flow from the upper tank to the loop portion; (ii) a first inlet connecting the upper tank to the upflow section of the loop section, the first inlet allowing fermentation liquor present in the loop section to flow from the loop section to the upper tank; (iii) a vent tube for venting exhaust gases from the upper vessel; and (iv) Visual inspection means wherein the fermentation reactor further comprises: (v) at least one inlet for supplying a substrate comprising an ammonium compound; and (vi) at least a sensor for determining the concentration of nitrate in the fermentation broth; Includes. [Brief description of the drawings]

[0014] Figure 1 shows that biomass production in the pilot plant (solid line) decreases over time, while nitrate production (dashed line) increases over time. This trend has been found in laboratory tests in both the pilot plant as well as the production plant.

[0015] The present invention will now be described in more detail below.

[0016] Detailed Description of the Invention Thus, the inventors of the present invention have found that because the nitrogen source provided to the fermentation process may act both as a nutrient for the growth of the microorganism, e.g., a methanotrophic microorganism, as well as a competitive inhibitor of methane consumption, e.g., by inhibiting the methane monooxygenase enzyme, the concentration of the nitrogen source needs to be adjusted and / or controlled to optimize biomass production of the methanotrophic microorganism, e.g., methylococcus capsulatus.

[0017] Methylococcus capsulatus is a novel 3 ) or ammonium (NH 4 + ) to nitrite (NO 2 - The enzyme required is methane monooxygenase (MMO), which can oxidize ammonia and methane and hydroxylamine oxidoreductase (HAO) via the following reaction. This reaction requires oxygen. [ka]

[0018] Without being bound by theory, the inventors of the present invention believe that in the first step (K1) of autotrophic nitrification, nitrite produced by methanotrophic microorganisms, such as methylococcus capsulatus, is oxidized to nitrate by nitrite oxidoreductase (NXR) following the following reaction: [ka]

[0019] The above reactions (K1 and K2) and the kinetics of the reversible reactions are considered in combination with the formation of nitrate substantially directly from ammonia with almost no trace of nitrite formation using methanotrophic microorganisms, such as M. capsulatus.

[0020] Surprisingly, the inventors of the present invention have found from their experiments that the supply of a nitrogen source, e.g. ammonia, to the fermentation of methanotrophic microorganisms, e.g. M. capsulatus, needs to be controlled and regulated in order to maintain the nitrate concentration below a certain level in order to avoid a reduction in biomass development and / or to provide high biomass development.

[0021] In the present context the term "high biomass development" relates to a biomass concentration of more than 1 g / l; such as more than 5 g / l; for example more than 10 g / l; for example more than 15 g / l; for example more than 20 g / l; such as more than 25 g / l; for example more than 30 g / l; for example more than 50 g / l; for example more than 70 g / l; for example in the range 1-100 g / l; for example in the range 5-90 g / l; for example in the range 10-80 g / l; for example in the range 20-70 g / l; for example in the range 30-65 g / l; for example in the range 40-60 g / l; for example in the range 45-55 g / l.

[0022] Thus, for example, nitrate formed in cultures by methanotrophic microorganisms, such as M. capsulatus, using ammonia as nitrogen source, may be used as a responsible indicator of fermentation stress, and the fermentation process can therefore be controlled by manipulating the nitrate concentration in the fermentation reactor, for example by reducing the flow of the nitrogen source or even stopping the flow to zero L / min.

[0023] The inventors have found that this method of controlling or regulating a fermentation process may be essential to ensure high productivity of methanotrophic biomass, such as M. capsulatus biomass, regardless of whether the fermentation process is run in batch, fed-batch or continuous mode.

[0024] This effect and the importance of control and / or regulation is demonstrated in the following experiments of both pilot scale and production / industrial laboratory tests.

[0025] Thus, surprisingly, the inventors of the present invention have found a fermentation method and a fermentation reactor in which the nitrogen source may be controlled and / or regulated to improve the production of methanotrophic biomass.

[0026] In a preferred embodiment of the present invention, the present invention relates to a fermentation method for fermenting a fermentation broth comprising at least one microorganism in a fermentation reactor, the fermentation method comprising the steps of: d) feeding the carbon substrate to a fermentation reactor such that the carbon substrate is dissolved or partially dissolved in the fermentation broth; e) feeding the nitrogen substrate to a fermentation reactor so that the nitrogen substrate is dissolved or partially dissolved in the fermentation broth; and f) maintaining the nitrate concentration of the fermentation broth at less than 0.035 g / l and / or maintaining the nitrate concentration of the fermentation broth at less than 0.01 g nitrate / g biomass wherein the at least one microorganism comprises at least one methanotrophic microorganism.

[0027] The nitrate concentration of the fermentation broth during fermentation may be maintained at less than 0.035 g / l; such as less than 0.033 g / l; for example less than 0.03 g / l; for example less than 0.028 g / l; for example less than 0.025 g / l; for example less than 0.022 g / l; for example less than 0.02 g / l; for example less than 0.018 g / l; for example less than 0.015 g / l; for example less than 0.01 g / l; for example less than 0.005 g / l; for example less than 0.01 g / l; for example 0 g / l.

[0028] In one embodiment of the invention, the nitrate concentration of the fermentation broth during fermentation is in the range of 0-0.035 g / l; for example in the range of 0.001-0.033 g / l; for example in the range of 0.002-0.03 g / l; for example in the range of 0.003-0.025 g / l; for example in the range of 0.004-0.02 g / l; for example in the range of 0.005-0.015 g / l; for example in the range of 0.007-0.01 g / l.

[0029] The nitrogen source may be a gaseous nitrogen substrate or an aqueous nitrogen substrate.

[0030] Preferably, the nitrogen source may be selected from ammonia; ammonium compounds; and / or molecular nitrogen. Even more preferably, the nitrogen source is ammonia.

[0031] The ammonium compound may be selected from ammonium carbonate, ammonium chloride, ammonium sulfate, ammonium hydroxide, and / or ammonium nitrate. Preferably, the ammonium compound is ammonium hydroxide.

[0032] In one embodiment of the invention, the nitrogen source may be provided to the fermentation broth at a concentration of less than 0.1 g / l; such as less than 0.09 g / l; for example less than 0.08 g / l; such as less than 0.07 g / l; for example less than 0.06 g / l; such as less than 0.05 g / l; for example less than 0.04 g / l; such as less than 0.03 g / l; for example less than 0.02 g / l; such as less than 0.01 g / l; for example less than 0.005 g / l; for example less than 0.001 g / l.

[0033] In a further embodiment of the invention, the nitrogen source may be supplied to the fermentation broth at a concentration in the range of 0.001-0.1 g / l; such as in the range of 0.005-0.09 g / l; for example in the range of 0.01-0.08 g / l; for example in the range of 0.02-0.075 g / l; for example in the range of 0.04-0.07 g / l; for example in the range of 0.05-0.06 g / l.

[0034] In a further embodiment of the invention, the nitrogen source fed to the fermentation reactor may not be a nitrate.

[0035] The nitrate concentration in the fermentation broth may depend on the biomass concentration.Therefore, in a preferred embodiment of the present invention, the nitrate concentration in the fermentation broth may be maintained at less than 0.01g nitrate / g biomass; for example, less than 0.008g nitrate / g biomass; for example, less than 0.006g nitrate / g biomass; for example, less than 0.004g nitrate / g biomass; for example, less than 0.002g nitrate / g biomass; for example, less than 0.001g nitrate / g biomass; for example, less than 0.0005g nitrate / g biomass; for example, 0g nitrate / g biomass.This calculation of the concentration of nitrate is based on the fermentation broth containing viable methanotrophic microorganisms.

[0036] Preferably, the carbon substrate may be a gaseous carbon substrate.

[0037] Preferably, the carbon substrate may be selected from alkanes, and preferably, the alkanes are C1 compounds. Even more preferably, the carbon substrate may be methane, methanol, natural gas, biogas, synthetic gas, or any combination thereof. Even more preferably, the carbon substrate may be methane.

[0038] As mentioned above, the carbon and / or nitrogen sources (as well as other components added to the fermentation broth) may be added as gases and these gases need to be dissolved in the fermentation broth, which may be an aqueous fermentation broth, so as to be available for the development of biomass and available to the microorganisms.

[0039] In general, there are industrial problems with mass transfer of substrates (e.g., carbon sources; and oxygen sources), and there is ongoing interest and effort in improving this mass transfer. One method of improving fermentation in a U-loop fermenter may be described in WO2010 / 06931 and / or WO2003 / 016460, which are incorporated herein by reference.

[0040] Thus, in the present invention, the term "dissolved or partially dissolved in the fermentation broth" relates to the problem known in the art of converting a gaseous substrate from a gas phase to an aqueous phase, which is available to at least one microorganism.

[0041] In a preferred embodiment of the invention, the nitrate concentration determined may be the dissolved nitrate concentration.

[0042] In further embodiments of the invention, the nitrate concentration of the fermentation broth may be determined by in-line analysis; by online analysis; or by offline or at-line analysis. Preferably, the nitrate concentration of the fermentation broth may be determined by in-line or online analysis.

[0043] In yet another embodiment of the invention, the nitrate concentration of the fermentation broth may be determined continuously by in-line or online analysis.

[0044] In the context of the present invention, the term "in-line analysis" relates to a sensor that may be placed in a process vessel or in a stream of flowing material to perform an analysis of one or more selected components.

[0045] In the context of the present invention, the term "online analysis" relates to a sensor that is connected to a method and may perform automatic sampling. An online analyzer may also be called an in-line analyzer.

[0046] Online analyzers and in-line analysis allow for continuous process control.

[0047] In the context of the present invention, the terms "off-line analysis" or "at-line analysis" may be used interchangeably and relate to sensors characterized by manual sampling followed by discontinuous sample preparation, measurement, and evaluation. Direct method control may not be possible since material properties can change between sampling and results.

[0048] In one embodiment of the invention, an oxygen substrate may be fed to the fermentation reactor. Preferably, the oxygen substrate may be dissolved or partially dissolved in the fermentation broth.

[0049] In a further embodiment of the invention, one or more nutrients; one or more pH adjusting components and / or water may be fed to the fermentation reactor. Preferably, the one or more nutrients; one or more pH adjusting components and / or water may be dissolved or partially dissolved in the fermentation broth.

[0050] The fermentation may be a batch, fed-batch or continuous fermentation. Preferably, the fermentation process is a continuous fermentation process.

[0051] Preferably, the methanotrophic organism may be a methanotrophic bacterium, such as Methylococcus capsulatus (used interchangeably with M. capsulatus).

[0052] The methanotrophic bacteria may be provided in a co-fermentation with one or more heterotrophic bacteria.

[0053] The following heterotrophic bacteria may be particularly useful for co-fermentation with M. capsulatus; Ralstonia spp.; Bacillus brevis; Brevibacillus agri; Alcaligenes acidovorans; Aneurinibacillus danicus and Bacillus firmus. Suitable yeasts may be selected from Saccharomyces and / or Candida species.

[0054] Preferred heterotrophic bacteria are selected from Alcaligenes acidovorans (NCIMB 13287), Aneurinibacillus danicus (NCIMB 13288) and Bacillus firmus (NCIMB 13289), and combinations thereof.

[0055] In one embodiment of the invention, the methanotrophic organism may be a genetically modified methanotrophic organism and / or the heterotrophic organism may be a genetically modified heterotrophic organism.

[0056] The fermentation reactor and / or fermentation process according to the invention may have particular relevance for the production of single cell proteins (SCP) by continuous culture fermentation processes, for example by Methylococcus capsulatus.

[0057] Preferred methanotrophic bacteria are species of the Methylococcus family, in particular Methylococcus capsulatus, which utilize methane or methanol as a carbon source and ammonia, nitrate or molecular nitrogen as a nitrogen source for protein synthesis.

[0058] A preferred embodiment of the present invention relates to a fermentation reactor comprising a loop section and an upper tank, said loop section comprising a downflow section connected to an upflow section via a U-section, the upper tank comprising: (i) a first outlet connecting the upper tank to the downflow portion of the loop portion and allowing the fermentation liquid present in the upper tank to flow from the upper tank to the loop portion; (ii) a first inlet connecting the upper tank to the upflow section of the loop section, the first inlet allowing fermentation liquor present in the loop section to flow from the loop section to the upper tank; (iii) a vent tube for venting exhaust gases from the upper vessel; and (iv) Visual inspection means wherein the fermentation reactor further comprises: (v) at least one inlet for supplying a substrate comprising an ammonium compound; and (vi) at least a sensor for determining the concentration of nitrate in the fermentation broth; Includes.

[0059] Preferably, the fermentation reactor may include at least one feed pump configured and / or controlled to automatically regulate the nitrate concentration in the fermentation broth.

[0060] In the present context, the term "regulating the nitrate concentration" refers to the action of either reducing the nitrate concentration in the fermentation broth or increasing the nitrate concentration in the fermentation broth. Preferably, the term "regulating the nitrate concentration" refers to the action of reducing the nitrate concentration.

[0061] In one embodiment of the invention, the nitrate concentration in the fermentation broth may be regulated by adjusting the flow of a nitrogen source to the fermentor; adjusting the flow of a carbon source to the fermentor; adjusting the flow of oxygen; adjusting the flow of nutrients; or a combination thereof.

[0062] The U-section of the loop reactor may connect the lower part of the downflow section to the lower part of the upflow section. Additionally, the upper part of the upflow section may be connected to a first inlet connecting the upper vessel to the upper part of the upflow section. A first outlet may connect the upper vessel to the upper part of the downflow section.

[0063] In the present context, the term "fermentation reactor" refers to a reactor comprising an upper tank connected at the upper end to a downflow section and an upflow section, the downflow section and the upflow section being connected at the lower end via a U-section.

[0064] In the present context, the term "loop reactor" relates to a particular example of a fermentation reactor.

[0065] The loop section of the present invention refers to the downflow section, the upflow section, and the connection section of the lower end of the upflow section and the lower end of the downflow section formed by the U-section. Thus, the "loop section" refers to a fermentation reactor without an upper vessel.

[0066] In the present context, the term "U-section" relates to a bend provided at the bottom of the fermentation reactor or loop reactor that connects the lower end of the upflow section and the lower end of the downflow section. Preferably, the upflow section and the downflow section are vertical or substantially vertical.

[0067] In the present context, the term "upper tank" relates to a vessel located at the top of the fermentation reactor and involved in the removal of effluent gas from the fermentation liquid. Preferably, the upper tank is only partially filled with fermentation liquid during operation / fermentation. In one embodiment of the present invention, the term "partially filled with fermentation liquid" relates to a ratio between fermentation liquid and gas of 90:10; such as a ratio of 80:20; such as a ratio of 70:30; such as a ratio of 60:40; such as a ratio of 50:50; such as a ratio of 40:60; such as a ratio of 30:70; such as a ratio of 20:80; such as a ratio of 10:90.

[0068] In the context of the present invention, "visual inspection means" relates to one or more means that allow a person skilled in the art to obtain direct information regarding the foaming properties of the upper tank.

[0069] In one embodiment of the present invention, the direct information may be real-time information regarding the foaming characteristics in the upper tank.

[0070] In a further embodiment of the present invention, the foaming characteristics in the upper tank may include foam density, foam height, and level of turbulence provided in the upper tank.

[0071] Turbulence in the upper tank may be provided to the fermentation liquid present in the upper tank as the fermentation liquid is forced from the upflow section through the first inlet into the upper tank.

[0072] Foam density may be an expression of the size of the air bubbles in the foam. The larger the air bubbles in the foam, the lower the foam density, kg foam / m 3 The smaller the bubbles in the foam, the higher the foam density, kg bubbles / m 3 becomes larger.

[0073] In one embodiment of the present invention, the visual inspection means may be arranged with a horizontal or substantially horizontal inspection view.

[0074] In a further embodiment of the invention, the visual inspection means may be positioned on the side of the upper vessel to allow a combined view above the surface of the fermentation liquid and below the surface of the fermentation liquid.

[0075] Preferably, the visual inspection means may be located at the edge of the upper reservoir.

[0076] Even more preferably, the visual inspection means may be located at an end of the upper tank providing a view from the first inlet (or upflow portion) towards the first outlet (or downflow portion).

[0077] In one embodiment of the present invention, the visual inspection means may be an inspection hole, a camera, or a combination of an inspection hole and a camera.

[0078] Preferably, the inspection hole may be a sight glass.

[0079] The camera may be an in-line camera.

[0080] In one embodiment of the present invention, the upper chamber may be provided with a light source to improve visual inspection within the upper chamber. The light source may be provided as a window that allows ambient light to enter the upper chamber and / or as an artificial light source integrated into the upper chamber.

[0081] In further embodiments of the invention, the light source may be provided as a separate feature within the sight glass (e.g., as a separate artificial light source) or as an integrated feature (e.g., as an integrated artificial light source).

[0082] In addition to visual inspection, the upper reservoir may be provided with at least one bubble sensor within the upper reservoir.

[0083] To avoid excessive foaming, an antifoam agent may be added to the fermentation liquor, and therefore the upper vessel may be provided with an antifoam inlet.

[0084] In an embodiment of the invention the fermentation reactor, preferably the loop portion, comprises an ion sensor or analyzer for determining the content of one or more ionic species in the fermentation broth, preferably the one or more ionic species are selected from phosphate, calcium, hydrogen, nitrate, nitrite and / or ammonium, preferably nitrate and / or nitrite.

[0085] In a further embodiment of the present invention, the loop reactor may be provided with a circulation pump.

[0086] Preferably, the circulation pump may be located in an upper half portion of the downward flow section.

[0087] In one embodiment of the invention, the fermentation reactor may include a flow reduction device. Preferably, the flow reduction device may be inserted upstream from the first inlet and in the upper half of the upflow section.

[0088] In further embodiments of the invention, preferably the loop portion of the fermentation reactor may comprise one or more gas inlets; one or more water inlets; and / or one or more fermentation medium inlets.

[0089] The gas inlet(s), water inlet(s), and / or fermentation medium inlet(s) may be computer controlled. Preferably, the gas inlet(s), water inlet(s), and / or fermentation medium inlet(s) may be computer controlled based on data obtained from one or more sensors or analyzers.

[0090] To provide improved fermentation conditions, the distribution of gaseous substrates, such as methane, in the fermentation broth may be important. Thus, the loop portion of the fermentation reactor may include one or more active devices for distributing gas in the fermentation broth.

[0091] In one embodiment of the present invention, the one or more active devices for distributing gas into the fermentation broth are micro- or nano-spargers for introducing and / or distributing gas into the fermentation broth; and / or dynamic motion devices, such as dynamic mixers, located in the loop portion of the reactor.

[0092] In addition to or as an alternative to a dynamic mixer, the loop section may include one or more inert mixing elements. In one embodiment of the invention, the one or more inert mixing elements may be static mixers.

[0093] In addition to the importance of proper degassing in the upper vessel, it may be important to improve mass transfer of the gaseous substrate into the liquid phase where the gas becomes available to the biocatalyst (e.g., methanotrophic organism) in an energy efficient manner.

[0094] Furthermore, as mentioned above, it may also be important to improve the efficiency of waste gas removal by improving the transfer of waste gas from the liquid phase to the gas phase for removal from the fermenter, which is preferably carried out in the upper tank.

[0095] Preferably, this improved efficiency in waste gas removal may be provided by operating the U portion of the loop section under increased pressure.

[0096] This improved mass transfer combined with improved gas removal in the upper vessel may be achieved in a fermentation reactor, a loop reactor, according to the present invention, comprising a loop section having a substantially vertical downflow section, a substantially vertical upflow section, and a U-section having a substantially horizontal connecting section, the U-section connecting the lower end of the downflow section with the lower end of the upflow section, wherein an upper vessel may be provided above the loop section and connecting the upper end of the downflow section with the upper end of the upflow section.

[0097] In one embodiment of the invention, the upper tank may have a diameter substantially larger than the diameter of the loop portion, the downflow portion, and / or the upflow portion.

[0098] In one embodiment of the invention, the U-section of the fermenter may comprise an outlet, preferably located in the U-section of the upper vessel or loop section of the fermentation reactor, for collecting the fermentation broth.

[0099] The fermentation reactor may include one or more gas injection points, which are located in the downflow section, the U section and / or the upflow section, depending on the desires and requirements. Preferably, the one or more gas injection points are located in the downflow section.

[0100] Immediately after the gas injection point(s), at least one active mixing element and / or at least one non-active mixing element for the dispersion of the gas(es) introduced into the fermentation liquid.

[0101] By increasing the pressure in the U-loop, loop reactor, increased mass transfer from the gas phase to the liquid phase may be improved. Thus, a first pressure control device may be inserted in the U-section of the fermenter to increase the pressure of at least a first zone of the U-section of the fermenter relative to the pressure of the second zone of the fermenter.

[0102] In a preferred embodiment of the present invention, the first pressure control device may be inserted at the upper end of the downward flow section, and the second pressure control device may be inserted downstream of the first pressure control device in terms of the U section of the fermenter and the flow direction of the fermentation liquid.

[0103] The first pressure control device may be a valve (e.g., a commercially available valve type), a pump, such as a propeller pump, a lobe pump, or a turbine pump, or the pressure may be increased by injection of pressurized air or another gas, such as an inert gas. Preferably, the first pressure control device is a propeller pump, which also creates a liquid circulation in the fermenter.

[0104] The second and optionally third pressure control devices may be located in the downflow section, the upflow section or the U section, but preferably the second pressure control device is located in the upper half of the upflow section. Preferably the third desired pressure control device is located in the upper half of the upflow section and upstream of the second pressure control device in the direction of flow of the fermentation liquid. The second and / or third pressure control device is selected from the group of devices including valves (e.g. commercial valve types), static mixers, hydrocyclones, pumps (e.g. propeller, lobe or turbine pumps), pressure control valves, plates with holes, nozzles or jets, or narrowing of the diameter or cross section of the fermenter section in which it is located.

[0105] In an embodiment of the invention, improved mass transfer of the gaseous substrate may be provided in the U section of the fermentation reactor according to the invention.

[0106] In a further embodiment of the invention, waste gas removal may be provided in the upper tank of the fermentation reactor according to the invention.

[0107] In one embodiment of the present invention, means are provided to allow flushing of the headspace to improve waste gas removal and reduce the risk of explosive gas mixtures forming in the headspace of the fermenter.

[0108] This flushing may be achieved by arranging gas flushing means in the upper vessel, e.g. a device for adding and / or removing gas in the head space. Preferably, the gas flushing means may be arranged above the liquid level to create a gas flow of flushing gas in parallel, simultaneous or cross-current to the liquid flow in the upper part of the fermenter. Gas adding means may also be arranged below the liquid level in the upper part. Alternatively or additionally, waste gas removal may be increased by reducing the pressure in the head space by applying suction or vacuum, thus reducing the pressure in the head space, and / or by installing flow modification means in the upper part. The invention also allows the energy applied to increase the pressure to be recovered for reuse. This may be achieved by connecting the second and optionally the third pressure control device to a brake or generator for reducing the pressure with the propeller pump. If a generator is connected to the second and / or the third pressure control device, part of the energy applied to the system may be collected, thus reducing the overall energy consumption of the system.

[0109] In the present context, the term "flushing" is used in reference to a method carried out in the upper vessel to remove or assist in the removal of effluent gases from the headspace of the upper vessel and / or the fermentation broth in the upper vessel.

[0110] The upper tank provided according to the invention may be designed to comprise between 1% and 99% of the total volume of the fermenter, but preferably between 10% and 60% of the total fermenter volume, and even more preferably between 40-50% of the total fermentation volume. In one embodiment of the invention, the volume of the upper tank may be less than the volume of the U-section.

[0111] The upper vessel may be provided with means for modifying the flow of liquid or gas to aid in mixing in the fermentation reactor or to aid in the release of gas bubbles from the fermentation liquid. The means for modifying the flow of gas or liquid may be dynamic mixers, baffles, or static mixers.

[0112] The size of the fermentor, both diameter and height, may vary depending on the needs of the total fermentor volume.

[0113] In one embodiment of the invention, the fermentation reactor according to the invention may be provided with a drive gas inlet into which a drive gas may be introduced to drive the carbon dioxide in the liquid phase into a separable exit gas phase. Preferably, the drive gas inlet may be located upstream of the upper tank and / or upstream of the first inlet.

[0114] The drive gas, i.e. the gas used to displace carbon dioxide from the dissolved phase (usually nitrogen, but optionally another inert, non-flammable gas), may be introduced, for example, at one or more points from the beginning of the substantially vertical upflow zone to the entry into the effluent gas removal zone, however, particularly preferably, it is introduced at one or more points between the top of the vertical portion of the upflow zone (e.g. the top 20%, more preferably the top 10%) and the start of the flattest (i.e. most horizontal) portion of the effluent zone.

[0115] In the context of the present invention, the term "drive gas" is used in reference to the process carried out at the top of the loop section, preferably the upflow section, which aids in the removal of effluent gases from the fermentation broth into the gas phase.

[0116] In one embodiment of the invention, the fermentation reactor includes both an inlet in the upper tank for introducing flushing gas into the upper tank and an inlet at the top end of the upflow section of the loop section for introducing a drive gas to move the effluent gas from the fermentation broth to the gas phase.

[0117] One advantage of the present invention may be that improved utilization of gaseous substances added to a fermentation reactor may be provided.

[0118] The productivity of the fermentation reactor and / or fermentation process according to the invention may be further optimized in that the circulating fermentation liquor experiences alternating pressures during circulation in the fermenter, with increased mass transfer and solubility of the substrate gas in the liquid phase in zones with increased pressure. Productivity may also be improved by release of gas, e.g. waste gas, from the circulating fermentation liquor, which release is increased in zones where pressure is reduced.

[0119] In one embodiment of the invention, the increased pressure in the loop portion of the fermentation reactor, in the first zone and / or between the first and second pressure controllers may be provided by applying a pressure of more than 0.5 bar above atmospheric pressure; such as a pressure of more than 1 bar above atmospheric pressure; for example a pressure of more than 1.5 bar above atmospheric pressure; for example a pressure of more than 2 bar above atmospheric pressure; for example a pressure of more than 2.5 bar above atmospheric pressure; for example a pressure of more than 3 bar above atmospheric pressure; for example a pressure of more than 3.5 bar above atmospheric pressure; for example a pressure of more than 4 bar above atmospheric pressure; for example a pressure of more than 4.5 bar above atmospheric pressure; for example a pressure of more than 5 bar above atmospheric pressure; for example a pressure of more than 5.5 bar above atmospheric pressure, such as a pressure of more than 6 bar above atmospheric pressure; for example a pressure of more than 7 bar above atmospheric pressure.

[0120] In another embodiment of the invention, the increased pressure in the loop portion of the fermentation reactor, in the first zone and / or between the first and second pressure controllers may be provided by applying a pressure in the range of 0.5 to 10 bar above atmospheric pressure; such as a pressure in the range of 1 to 9 bar above atmospheric pressure; such as a pressure in the range of 1.5 to 8 bar above atmospheric pressure; such as a pressure in the range of 2 to 7 bar above atmospheric pressure; such as a pressure in the range of 3 to 6 bar above atmospheric pressure; such as a pressure in the range of 4 to 5 bar above atmospheric pressure.

[0121] In yet another embodiment of the invention, the pressure in the upper chamber may be less than 0.5 bar above atmospheric pressure; such as 0.25 bar above atmospheric pressure; such as 0.1 bar above atmospheric pressure; such as about atmospheric pressure; such as less than 0.75 bar below atmospheric pressure; such as 0.5 bar below atmospheric pressure; such as less than 0.25 bar below atmospheric pressure; such as 0.1 bar below atmospheric pressure.

[0122] Further details regarding suitable modifications to the loop reactor and features regarding how to run such a loop reactor, and processing of the resulting biomass, may be as described in WO2010 / 069313; WO2000 / 70014; WO2003 / 016460; WO2018 / 158319; WO2018 / 158322; WO2018 / 115042 and WO2017 / 080987, all of which are incorporated by reference.

[0123] Examples of suitable downstream processing of the obtained biomass to provide various fractions may be as described in WO2018 / 115042.

[0124] Sensors may include biosensors, electrochemical sensors, e.g., ion-sensitive electrodes or sensors based on FIA (flow injection analysis) and optical measurements, e.g., spectrophotometric devices. Near-infrared (NIR) probes may also be used to measure the concentration of several different components, e.g., cells, amino acids, methanol, ethanol and / or different ions, within the broth or cells of the fermenter. Fermentation reactors also measure gaseous and volatile components in the headspace (e.g., CO 2 and / or C.H. 4The fermentation reactor may be equipped with a mass spectrometry (MS) sensor or electronic nose to determine the concentration of . The MS sensor or electronic nose may control the pressure applied to the fermenter and / or the addition of gaseous components, such as methane and / or air / oxygen, and / or the addition of gaseous ammonia or ammonia / ammonium in solution. To determine the gas bubble size in the broth, a high speed camera may be installed in the U-section of the fermentation reactor, preferably in conjunction with the gas injection. The bubble size may be determined by image processing of the data from the high speed camera.

[0125] Typically, the fermentation reactor according to the invention may be run in a continuous operation mode after cleaning and sterilization procedures, followed by a start-up period during which water, necessary nutrients and microorganisms are added to the fermentation reactor. The fermentation broth may be circulated in the fermentation reactor mainly by a first pressure control device. The addition of gaseous substrate may then be started and fermentation may be initiated. When the density of the microorganisms reaches a concentration of approximately 0.5-10%, preferably 1-5% (by dry weight), the fermentation broth may be continuously withdrawn from the fermentation reactor, for example from the upper tank or U-part, and subjected to downstream processing, for example as described in WO2018 / 115042.

[0126] Harvesting of the fermentation broth may be initiated simultaneously with make-up water, addition of aqueous substrate, and / or recirculation of the supernatant at a dilution rate, depending on the microorganism used in the fermentation. The harvested broth and the substrate components in solution as a make-up of the substrate gas, addition of additional water, recirculation of the supernatant may be controlled by a computer receiving data from gas sensors and appropriate calculations to provide the required amount of each component to obtain optimized growth of the organism.

[0127] In one embodiment of the present invention, the fermentation process and fermentation reactor may be a laboratory scale, a pilot plant and / or a production or industrial plant. Preferably, the fermentation process and fermentation reactor may be a production or industrial plant.

[0128] It should be noted that embodiments and features described in the context of one aspect of the invention also apply to other aspects of the invention.

[0129] All patent and non-patent references cited in this application are hereby incorporated by reference in their entirety.

[0130] The invention will now be described in further detail in the following non-limiting examples. The present disclosure relates, for example, to the following: [Section 1] A fermentation process for fermenting a fermentation broth comprising at least one microorganism in a fermentation reactor, the fermentation process comprising the steps of: a) feeding a carbon substrate to a fermentation reactor such that the carbon substrate is dissolved or partially dissolved in the fermentation broth; b) feeding the nitrogen substrate to a fermentation reactor so that the nitrogen substrate is dissolved or partially dissolved in the fermentation broth; and c) maintaining the nitrate concentration of the fermentation broth at less than 0.035 g / l and / or maintaining the nitrate concentration of the fermentation broth at less than 0.01 g nitrate / g biomass wherein the at least one microorganism comprises at least one methanotrophic microorganism. [Section 2] Item 2. The fermentation method according to item 1, wherein the nitrate concentration of the fermentation broth during fermentation is in the range of 0 to 0.035 g / l; for example, in the range of 0.001 to 0.033 g / l; for example, in the range of 0.002 to 0.03 g / l; for example, in the range of 0.003 to 0.025 g / l; for example, in the range of 0.004 to 0.02 g / l; for example, in the range of 0.005 to 0.015 g / l; for example, in the range of 0.007 to 0.01 g / l. [Section 3] Item 3. The fermentation method according to any one of Items 1 to 2, wherein the nitrogen source is selected from ammonia; an ammonium compound; and / or molecular nitrogen, and preferably the nitrogen source is ammonia. [Section 4] The fermentation method according to any one of Items 1 to 3, wherein the fermentation is batch fermentation, fed-batch fermentation or continuous fermentation, and preferably the fermentation method is a continuous fermentation method. [Section 5] A fermentation reactor comprising a loop section and an upper tank, the loop section comprising a downflow section connected to an upflow section via a U-section, the upper tank comprising: (i) a first outlet connecting the upper tank to the downflow portion of the loop portion and allowing the fermentation liquid present in the upper tank to flow from the upper tank to the loop portion; (ii) a first inlet connecting the upper tank to the upflow section of the loop section, the first inlet allowing fermentation liquor present in the loop section to flow from the loop section to the upper tank; (iii) a vent tube for venting exhaust gases from the upper vessel; and (iv) Visual inspection means wherein the fermentation reactor further comprises: (v) at least one inlet for supplying a substrate comprising an ammonium compound; and (vi) at least a sensor for determining the concentration of nitrate in the fermentation broth; A fermentation reactor comprising: [Section 6] Item 6. The fermentation reactor of item 5, wherein the fermentation reactor comprises at least one feed pump configured and / or controlled to automatically regulate the nitrate concentration in the fermentation broth. [Section 7] Item 7. The fermentation reactor according to any one of Items 5 to 6, wherein the fermentation reactor is for fermentation of a methanotrophic organism. [Section 8] The fermentation reactor according to any one of items 5 to 7, further comprising an ion sensor or analyzer for determining the content of one or more ion species in the fermentation broth, preferably the one or more ion species being selected from phosphate, calcium, hydrogen, nitrite and / or ammonium. [Section 9] The fermentation reactor according to any one of items 5 to 8, wherein the loop portion of the fermentation reactor includes one or more gas inlets; one or more water inlets; and / or one or more fermentation medium inlets. [Section 10] 10. The fermentation reactor of claim 9, wherein the one or more gas inlets; one or more water inlets; and / or one or more fermentation medium inlets are computer controlled based on data obtained from one or more sensors or analyzers.

[0131] Working Example Example 1 This example demonstrates the correlation between nitrate concentration in the fermentation broth and biomass development.

[0132] Nitrate formation was determined during the cultivation of M. capsulatus in a 1 L BIOSAT® B-Plus bioreactor (Sartorius, DK) where cooling jacket water flow, motor frequency, and 2M H 2 SO 4 Or an internal control loop regulating the dosage of 2M NaOH keeps the temperature at 42°C and the agitation at 10 RPS. -1 (rounds per second) and pH were maintained at 6.7 ± 0.05. Dissolved oxygen (DO) was maintained using a VisiFerm DO ECS 120 H 2 It was monitored using an optical DO electrode (Hamilton, USA).

[0133] Bioreactor, 96.81 g h -1 of sterile air and 4.95g·h -1 Sterilized methane (Instrument methane 3.5, AGA, DK) was continuously sparged.

[0134] The cultivation of M. capsulatus was initiated as a batch phase in 2NMS medium (nitrate mineral salt medium) and continued at stationary state (continuous phase fermentation) in AMS medium (ammonium mineral salt medium) when nitrate was exhausted. The feed flow rate during continuous cultivation was 48.95 10 -3 Lh -1 The cultures were allowed to reach stationary state before attempts to induce cometabolism were initiated.

[0135] Different steady-state ammonia pulse experiments were carried out in 1 L fermenters under fixed conditions where the biomass before and after the effect of the pulse along with the nitrate concentration were determined.

[0136] result Tables 1 and 2 below show that as a result of the pulse injection, the formation of nitrate increases with increasing ammonia concentration. The same experiment is maintained for 24 hours, and as the concentration of the ammonia pulse increases, the biomass suddenly decreases, approaching a wash out phase while the nitrate remains in the reactor.

[0137] Tables 1 and 2: Different ammonia concentrations were fed to a 1 L reactor at steady state and ammonia, nitrate, and biomass concentrations were measured before ammonia injection and at two different time points: 2 h after the pulse (Table 1) and 24 h after the pulse (Table 2). [Table 1] [Table 2]

[0138] The regulation of this high concentration of nitrogen source in the fermentation broth can be achieved by controlling the flow rate of the substrate to control the process so that nitrate is not formed, as well as the accumulation of nitrite and / or nitrate. During these controlled conditions, excess nitrate may be consumed by M. capsulatus and the nitrogen concentration of the fermentation broth may be reduced.

[0139] The same trend (excessive nitrate production leading to reduced biomass) is observed in the pilot plant as shown in Figure 1. Figure 1 shows that biomass production decreases with increasing nitrate production in the pilot plant. Similar trends are observed in the production plant and in the laboratory as discussed in Tables 1 and 2.

[0140] reference TIFF0007611174000005.tif65132

Claims

1. A continuous fermentation process for fermenting a fermentation broth comprising at least one microorganism in a fermentation reactor, the fermentation process comprising the steps of: a) feeding a carbon substrate to a fermentation reactor such that the carbon substrate is dissolved or partially dissolved in the fermentation broth; b) feeding the nitrogen substrate to a fermentation reactor so that the nitrogen substrate is dissolved or partially dissolved in the fermentation broth; and c) maintaining the nitrate concentration of the fermentation broth at less than 0.035 g / l and / or maintaining the nitrate concentration of the fermentation broth at less than 0.01 g nitrate / g dry weight biomass. Including, wherein the at least one microorganism comprises at least one methanotrophic microorganism, and the nitrate concentration in the fermentation broth is regulated by: regulating the flow of nitrogen substrate to the fermentation reactor; regulating the flow of carbon substrate to the fermentation reactor; adjusting the flow of oxygen; adjusting the flow of nutrients; or a combination thereof. Fermentation method.

2. The fermentation method according to claim 1, wherein the nitrate concentration of the fermentation broth during fermentation is in the range of 0 to 0.035 g / l.

3. The fermentation method according to any one of claims 1 to 2, wherein the nitrogen source is selected from ammonia; ammonium compounds; and / or molecular nitrogen.

4. A fermentation method described in any one of claims 1 to 3, wherein the nitrate concentration in the fermentation broth is adjusted by adjusting or stopping the flow of nitrogen substrate to the fermentation reactor.

5. A fermentation method as claimed in any one of claims 1 to 4, wherein the nitrate concentration of the fermentation broth is determined by in-line analysis; by online analysis; or by offline or at-line analysis.

6. A fermentation method described in any one of claims 1 to 5, wherein the nitrate concentration of the fermentation broth is determined by in-line or online analysis.

7. A fermentation method according to claim 1, wherein the oxygen substrate is supplied to a fermentation reactor and dissolved or partially dissolved in the fermentation broth.

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